The present invention relates to the field of epidermal repair. More particularly, the invention concerns the use of a molecule able to activate a heteromeric receptor comprising OSMRβ as a subunit, for the preparation of a composition for enhancing in vitro and/or in vivo keratinocyte migration.
The skin is a large and complex tissue providing a protective interface between an organism and its environment. Epidermis forms its external surface, and is mainly constituted of multiple layers of specialized epithelial cells named keratinocytes. Skin can be injured by many different causes, including micro-organisms, chemicals, behaviours, physical injury, ageing, U.V. irradiation, cancer, autoimmune or inflammatory diseases.
Epidermis homeostasis is regulated by a balance between differentiation and proliferation of keratinocytes, differentiating from the basal to the cornified layers of the skin. In response to epidermal stress or in some skin diseases, this equilibrium is broken. Keratinocytes become able to differentially respond to soluble mediators such as Epidermal Growth Factor (EGF) family members, and to additional growth factors and cytokines (FGFs, IGF-1, PDGF, HGF, TGFβ family members, GM-CSF, TSLP, IL-1, TNF-α). These modulators are produced by the keratinocytes themselves, the skin fibroblasts, the Langerhans cells or by immune infiltrating cells such as T lymphocytes. In response, the keratinocytes release additional signaling molecules, modulate the expression level of cell surface receptors, modify their cytoskeleton morphology, and modulate their migration, differentiation and proliferation capacities. These changes are associated with an inflammatory response, leading to either wound healing or to a chronic disease.
The cytokines of the IL-6 family are multifunctional proteins regulating cell growth and differentiation in a large number of biological systems, such as immunity, hematopoiesis, neural development, reproduction, bone modeling and inflammatory processes. This cytokine family encompasses nine different members: IL-6, IL-11, IL-27, leukemia inhibitory factor (LIF), cardiotrophin-1, cardiotrophin-like factor, ciliary neurotrophic factor, neuropoietin, and oncostatin M (OSM). The activities of theses cytokines are mediated through ligand-induced oligomerization of a dimeric or trimeric receptor complex. The IL-6 family of cytokines shares the gp130 receptor subunit in the formation of their respective heteromeric receptors (Taga and Kishimoto 1997). A recently described cytokine, named IL-31, has been classified by Dillon et al as a novel member of the gp130-IL6 family, because its receptor is a heterodimer comprising gp130-like type I cytokine receptor (GPL) and an OSMR subunit (Dillon, Sprecher et al. 2004).
Different publications have reported that some members of the IL-6 family may be implicated in certain skin diseases and wound healing processes. IL-6, IL-11, LIF and OSM have been found to be increased in psoriatic lesions (Bonifati, Mussi et al. 1998), and IL-6 and LIF are produced by purified keratinocytes (Paglia, Kondo et al. 1996; Sugawara, Gallucci et al. 2001). An impaired wound healing process has been reported in IL-6 and STAT3 deficient mice (Sano, Itami et al. 1999; Gallucci, Simeonova et al. 2000). However, further studies on cultured keratinocytes isolated from IL-6 deficient mice showed that the action of IL-6 on keratinocyte migration is mediated by dermal fibroblasts. Indeed, IL-6 alone did not significantly modulate the proliferation or migration of said IL-6-deficient keratinocytes, whereas IL-6 significantly induced their migration when co-cultured with dermal fibroblasts (Gallucci, Sloan et al. 2004).
OSM is secreted from activated T cells, monocytes stimulated by cytokines and from dendritic cells. OSM is a pro-inflammatory mediator, which strongly triggers protein synthesis in hepatocytes (Benigni, Fantuzzi et al. 1996). In humans, OSM and LIF display overlapping biological functions in a number of tissues by increasing growth regulation, differentiation, gene expression, cell survival. OSM is also known to elicit some unique biological functions, not shared with LIF, such as growth inhibition of some tumor cell lines or stimulation of AIDS-associated Kaposi's sarcoma cells. These shared and specific functions of OSM are explained by the existence of two types of OSM receptor complexes. Beside the common LIF/OSM receptor complex made of gp130/LIFRβ subunits, OSM is also able to specifically recognize a type II receptor associating gp130 with OSMRβ (also referred to as “OSMR” or “OSM-R”), which is expressed by endothelial cells, hepatic cells, lung cells, fibroblasts, hematopoietic cells and by some tumor cell lines. The subsequent signaling cascade involves activation of the Janus kinase (JAK 1, JAK 2, Tyk 2), followed by an activation of the Signal Transducer and Activator of Transcription (STAT1, STAT3) and of the Map kinase pathways.
In addition to its anti-neoplastic activity and its role in the pro-inflammatory response (Wahl and Wallace 2001); Shoyab et al, U.S. Pat. No. 5,451,506; Richards et al., U.S. Pat. No. 5,744,442), OSM has been described as stimulating the growth of dermal fibroblasts via a MAP kinase-dependent pathway, thereby promoting dermal wound healing (Ihn and Tamaki 2000).
Other cytokines are also known to have an effect on dermis. For example, Dillon et al (supra) suggest that overexpression of IL-31 may be involved in promoting the dermatitis and epithelial responses that characterize allergic and non-allergic diseases. These authors do not suggest to use IL-31 for promoting skin repair.
When skin is injured, its complete repair implies that both the dermis and the epidermis are repaired. Healing of epidermis and dermis, which comprise different cell-types, involve different mechanisms.
Currently, treatments for improving skin healing mainly target the dermis. However, epidermis reconstitution is necessary for a complete recovering. In some cases, for example in the case of large burns, ulcers or bedscores, physiological epidermal healing processes are not efficient enough for restoring the protecting function of skin. In such cases, it is necessary to rapidly cover the damaged area, to avoid infections and possibly dehydration. It is also necessary to stimulate epidermis regeneration. In the case of severe burns on less than half of the body surface, skin auto-graft is performed after excision of the burnt skin. To that aim, sane skin is taken from the patient and mechanically treated for increasing its surface. This “wick-skin” is then grafted on the lesions. When the burnt surface is too large (more than half of the body surface), this process is not feasible. It is then necessary to temporarily cover the wounds to avoid dehydration and infection. This is currently performed with either skin from cadavers, or with skin substitutes such as acellular dressings like tulle gras, possibly incorporating growth factors for improving wound healing. Examples of such skin substitutes are described in U.S. Pat. No. 6,132,759 or in WO 01/41820. In parallel, skin cells from the patient are expanded in vitro, in order to obtain epithelial layers that are then grafted. One to 2 m2 can be obtained in 3 weeks, from a few cm2 of sane skin. However, these techniques are long, costly, and need a heavy infrastructure to be successfully performed. Hence it is clear that there exists a real need for novel dermatological approaches for improving epidermal repair. Enhancing centripetal migration of the keratinocytes would clearly accelerate/enable healing and re-epithelialization. Acting on the keratinocytes' differentiation differentiation and migration is also necessary for treating specific diseases such as bullous epidermolysis. The phrase “bullous emolysis” designates a number of dermatitis of different origins (such as bleds, burns, autoimmune diseases, . . . ), leading to a detachment of the epidermis and liquid accumulation between dermis and epidermis. A particular example of bullous epidermolysis is bullous phemphigoid.
Improving epidermal repair is also important in the cosmetic field, where no efficient compositions exist for improving the aspect of scars, originating either from recent small wounds or from old cuts, spots, stretch marks and the like.
In this context, the inventors found that several cytokines, in particular OSM and IL-31, can enhance the migration of keratinocytes. Interestingly, these two cytokines bind to different heteromeric receptors, that both comprise OSMRβ as a subunit. The inventors have shown that normal human epidermal keratinocytes express gp130, GPL and OSMRβ.
As disclosed in the experimental examples below, OSM recruits the STAT3 signaling pathways, as well as the MAP kinase pathways in human epidermal keratinocytes. OSM up-regulates the expression of pro-inflammatory genes in these cells, including chemokines, defensin and the psoriasin. OSM also increases the thickness of reconstituted human epidermis and down-regulates a set of differentiation antigens. Interestingly, other cytokines, especially IL-17 and TNFα, act synergistically with OSM and potentiate its effects.
Experiments conducted by the inventors also revealed that IL-31 can mediate keratinocyte migration. The inventors however observed, in glioblastoma and melanoma tumor cells, that the action of IL-31 depends on the type of GPL subunit involved with OSMRβ in the formation of the heteromeric receptor. In particular, they noticed that a short form of GPL receptor exerts a profound inhibitory effect on the signaling of IL-31 and behaves as a dominant negative receptor.
Taken together, these results indicate that OSM and IL-31 play important roles in wound healing and other processes involving keratinocyte migration. These effects require the signal transduction involving the OSMRβ subunit in heteromeric receptors.